The aluminium-[18F]fluoride revolution: simple radiochemistry with a big impact for radiolabelled biomolecules

The aluminium-[18F]fluoride ([18F]AlF) radiolabelling method combines the favourable decay characteristics of fluorine-18 with the convenience and familiarity of metal-based radiochemistry and has been used to parallel gallium-68 radiopharmaceutical developments. As such, the [18F]AlF method is popular and widely implemented in the development of radiopharmaceuticals for the clinic. In this review, we capture the current status of [18F]AlF-based technology and reflect upon its impact on nuclear medicine, as well as offering our perspective on what the future holds for this unique radiolabelling method.


Fig. 1 A schematic of general PET radiolabelling methods
and may be considered as a parameter for optimisation when developing new [ 18 F]AlF labelling protocols (Kersemans et al. 2018) 1 Sep-Pak light QMA -0.9% NaCl (1 mL) Jiang et al. (2021) 2 Sep-Pak Accell Plus QMA Water (5 mL) 0.05 M NaOAc, pH 4.5 (300 µL) Lütje et al. (2019) 3 Sep-Pak light QMA Water 0.5 M NaOAc, pH 4.5 (500 µL) Giglio et al. (2018) 4 Sep-Pak Accell Plus QMA Purged to dryness 0.5 M NaHCO 3 or NaNO 3 or NaCl or NaOAc (600 µL) Kersemans et al. (2018) 5 Sep-Pak Accell Plus QMA light Water (6 mL) 0.9% NaCl (250 µL) Tshibangu et al. (2020) Ga, the relatively high temperatures (100-120 °C) required to chelate [ 18 F]AlF 2+ in N 3 O 2 configured chelators is not problematic as small peptides have been proven withstand these temperatures. However, large proteins where biological activity is derived from a precise tertiary structure can denature at these temperatures, which has encouraged the development of [ 18 F]AlF chelators for ambient temperature radiolabelling. Cleeren et al. (2016) described two promising acyclic chelators (Fig. 3, 5 and 6 (Cleeren et al. 2016). This was improved by the N 2 O 3 configured derivative (±)-H 3 RESCA (Fig. 3, 7) which utilised a cyclohexyl moiety to impart structural rigidity into the molecule (Cleeren et al. 2017). (±)-H 3 RESCA was radiolabelled at room temperature and was stable in rat plasma for at least 4 h, comparable to NODA derivatives. [ 18 F] Atom colours: carbon = light grey, hydrogen = white, oxygen = red, nitrogen = blue, fluorine = yellow, aluminium = dark grey. RT = room temperature. *Optional 1:1 (v/v) co-solvent included in the reaction mixture to improve RCY Page 5 of 28 Archibald and Allott EJNMMI radiopharm. chem. (2021) 6:30 around the 2-aminomethylpiperidine (AMP) group (Fig. 3, 8-10). All chelators radiolabelled efficiently at room temperature and pH 5 (RCY: 55-81%) with 2-AMPTA-HB (10) showing the greatest stability at 240 min post radiosynthesis in human serum (87 ± 5%), PBS (93 ± 1%) and saline (92 ± 2%). The in vivo evaluation of [ 18 F]AlF-2-AMPDA-HB showed low bone uptake at 2 h p.i. (1.63 ± 0.73%ID/g) (Russelli et al. 2020). The synthesis of a bifunctional derivative is now underway. These new chelators provide an elegant solution to radiolabelling heat-sensitive biomolecules and will benefit from a full evaluation in the clinic; we predict that this will be achieved within the next five years. It is our opinion that the commercial availability and affordability of pentadentate NODA and NOTA derivatives mean they are unlikely to be replaced by acyclic chelators, at least for the time being, for instances where ambient temperature radiochemistry is a convenience rather than a necessity. If the research community adopt these chelators (or future derivatives) for use in projects, their commercialisation may be expedited. The development of ambient temperature kit-based production protocols, akin to 68 Ga-trishydroxypyridinone (THP) which aims to be simple to use in the radiopharmacy setting (Young et al. 2017), may also increase the demand and implementation of alternative chelators. Nevertheless, it is important to remember that multistep synthesise of chelators can present a barrier to their use in projects which aim to focus on radioconjugate development.

[ 18 F]AlF-based radiopharmaceuticals
A range of [ 18 F]AlF-based radiopharmaceuticals have been developed for variety of biological targets and some have transitioned into the clinic for evaluation in patients. A selection of prominent examples is discussed in this review and their production scale, radiochemical yield (RCY), molar activity (A m ), status of automated radiosynthesis and clinical evaluation are summarised in Table 2; their structures, drawn in full where appropriate, are included in the relevant sections.
The clinical evaluation of [ 18 F]AlF-NOTA-octreotide in three healthy volunteers and 22 patients with neuroendocrine neoplasms (NEN) was well tolerated and in patients with NEN, high tumour uptake and tumour-to-background ratios were observed (Long et al. 2019 (Hou et al. 2020). All studies taken together, [ 18 F]AlF-NOTA-octreotide appears to be a promising alternative to [ 68 Ga]Ga-DOTA-TATE and has the potential for centralised, to increase availability and lower costs. All clinical trials of [ 18 F]AlF-NOTA-octreotide are presented in Table 3.

Imaging the immune system
Immune checkpoint inhibitors are an important therapeutic intervention for tumours evading the immune system, but patient response is variable (Postow et al. 2015;Royal Marrone et al. 2016). Immune checkpoints PD-1 and CTLA-4 are often targeted in combination to improve response rates but can lead to toxicity and immunerelated side effects (Khoja et al. 2017;Cousin and Italiano 2016). Radiopharmaceuticals to measure treatment response are of great interest and the [ 18 F]AlF method has been used to develop radioconjugates for imaging T-cell activation. Granzyme B is a serine protease released upon the activation of cytotoxic T cells and has been targeted by a gallium-68 labelled peptide [ 68 Ga]Ga-NOTA-GZP ( Fig. 6A) (Larimer et al. 2017). Preclinical imaging distinguished between responders and nonresponders to monotherapy (anti-PD-1) and combination immunotherapy (anti-PD-1 and anti-CTLA-4) with excellent predictive ability (Larimer et al. 2017;Goggi et al. 2020b). Goggi et al. (2020a) radiolabelled the peptide with the [ 18 F]AlF complex to improve PET sensitivity and spatial resolution. Formulated [ 18 F]AlF-mNOTA-GZP was produced in 50 min from [ 18 F]fluoride in a 17-25% RCY (n.d.c) and a molar activity of 45-90 GBq/μmol. The enzyme inhibition efficiency of both 68 Ga and [ 18 F]AlF peptides were similar. The uptake of [ 18 F]AlF-mNOTA-GZP correlated with changes in T cell populations and distinguished responders and non-responders to monotherapy and combination immunotherapy (Goggi et al. 2020a). An automated radiosynthesis of [ 18 F] AlF-mNOTA-GZP has not yet been described.
The interleukin-2 receptor (IL2R) is overexpressed on activated T cells and PET radioligands have been developed using recombinant IL2 as a targeting molecule. Gialleonardo et al. (2012)  [ 18 F]AlF-RESCA-IL2 showed high in vitro uptake in activated peripheral blood mononuclear cells (PBMC), and in vivo uptake in PBMC xenografts and lymphoid tissue, supporting further evaluation of this radioligand.

Imaging fibroblast activation protein (FAP)
Fibroblast activation protein (FAP) is highly expressed in many human cancers and can be targeted by quinoline-based FAP inhibitors (FAPIs) (Brennen et al. 2012;Jansen et al. 2013). Gallium-68 FAP radioligands have been evaluated and exhibit excellent tumour contrast Giesel et al. 2019). Given the promise for a radiolabelled   7A). [ 18 F]AlF-NOTA-FAPI-74 was evaluated in 10 patients with lung cancer and showed high contrast and low radiation burden (Giesel et al. 2021). This example highlights the synergy between 68 Ga and [ 18 F]AlF radioconjugates, and the simplicity in transforming from one isotope to another if an appropriate chelator has been employed in the conjugate. It is important to recognise the importance of chelator selection in developing gallium-68 radioconjugates, which is elegantly illustrated with FAPI-74, whereby thinking forward to potential future scalability beyond gallium-68 encouraged the use of the NOTA chelator; had DOTA or any other gallium-68 specific chelator being selected, then exploring the [ 18 F] AlF method would have been non-trivial.

Imaging the epidermal growth factor (EGF) receptor family
The epidermal growth factor (EGF) receptor family are overexpressed in many cancers and therapeutics have been developed towards these targets, including Cetuximab targeting EGFR and Trastuzumab targeting HER2 (Wang 2017;Xu et al. 2017b;Pernas and Tolaney 2019). Stratifying patients based on EGF receptor expression using PET imaging is of great interest and while zirconium-89 labelled monoclonal antibodies (mAb) with long-lived radioisotopes (t 1/2 = 3.3 days) have been developed, smaller biomolecule fragments with fast PK and radiolabelled short half-life isotopes are of great interest (Tolmachev and Orlova 2020).
The first Affibody molecule to be radiolabelled with the [ 18 F]AlF complex was Z HER2:2395 which targeted the HER2 receptor and showed specific tumour uptake (T:B of 7.4 ± 1.8 and tumour uptake of 4.4 ± 0.8%ID/g at 1 h p.i.) in SK-OV-3 xenografts (Heskamp et al. 2012). A NOTA-maleimide chelator was conjugated to the unique cystine residue of the Affibody molecule and radiolabelled within 30 min to produce [ 18 F]AlF-NOTA-Z HER2:2395 in a RCY of 21.0 ± 5.7% and molar activity of 7.7 ± 3.0 GBq/µmol; the affinity of the radioconjugate for HER2 was K d = 6.2 nM (Heskamp et al. 2012 (Xu et al. 2017a;Glaser et al. 2013).
A HER3 targeted affibody molecule Z HER3:8698 was radiolabelled with the [ 18 F]AlF complex using two methods: (1) direct [ 18 F]AlF chelation with NOTA-Z HER3:8698 and (2) a prosthetic group approach using inverse electron demand Diels-Alder (IEDDA) chemistry (Pieve et al. 2016). Approach 1 produced [ 18 F]AlF-NOTA-Z HER3:8698 in a RCY of 9.9-27.4% (n.d.c) and molar activity of 6.0-11.9 GBq/µmol (Fig. 8A). In approach 2, a NODA conjugated tetrazine (NODA-Tz) was synthesised alongside a TCO-conjugated  EGFR:1907 has been developed and produced in a radiochemical yield of 15% (Su et al. 2014). Despite implementing the [ 18 F]AlF method in the development of numerous Affibody molecule radioconjugates targeting the EGF family, the clinical translation of these probes is yet to be reported. However, given that 68 Ga-labelled Affibody molecules targeting HER2 have been evaluated in phase I/II clinical trials, we are likely to see [ 18 F]AlF derivatives in the near future (Sandström et al. 2016;Sörensen et al. 2016).

Imaging integrins
The integrins are a family of transmembrane receptors of which ⍺ v β 3 is involved in tumorigenesis and metastasis, making it an excellent target for PET imaging (Hamidi and Ivaska 2018). An arginine-glycine-aspartic acid (RGD) peptide sequence binds to integrins and forms the basis of many PET probes for imaging ⍺ v β 3 . One of the first [ 18 F] AlF-based RGD radioligand was reported by Liu et al. (2011) using a NOTA conjugated dimeric cyclic RGD peptide E[c(RGDyK)] 2 (NOTA-RGD 2 ). The [ 18 F]AlF-NOTA-RGD 2 peptide was produced in a RCY of 17.9% (d.c.) and molar activity of 11.1-14.8 GBq/ µmol and showed high tumour uptake (5.3 ± 1.7%ID/g) and good T:M contrast at 60 min p.i. (Fig. 9) (Liu et al. 2011 linker strategies aimed to optimise radiolabelling and PK performance of the probes and compared these to [ 18 F]AlF-NOTA-RGD 2 . All probes were synthesised in a RCY of 40-60%, with molar activities ranging from 14.8 to 37 GBq/µmol. All three probes exhibited favourable in vivo performance with high tumour uptake and good target-to-background ratios, but [ 18 F]AlF-NOTA-E[PEG 4 -c(RGDfK)] 2 (also known as 18 F-Alfatide II) was highlighted as a promising candidate for clinical translation owed to the lowest liver uptake and highest tumour uptake (2.92 ± 0.40%ID/g) (Guo et al. 2014). First-in-human studies of 18 F-Alfatide II commenced in 2015 in healthy volunteers and patients with brain metastases; the radioconjugate was well tolerated in all healthy volunteers and successfully visualised all 20 brain lesions

Current and future perspectives on the [ 18 F]AlF method
The [ 18 F]AlF method conveniently combines the favourable decay characteristics of fluorine-18 with the convenience of metal-based radiochemistry, as highlighted throughout this review. The avid adoption, implementation and continual development of the [ 18 F]AlF method is a testament to the positive contribution it has made to  Page 19 of 28 Archibald and Allott EJNMMI radiopharm. chem.

A "complex" relationship with gallium-68
Generator-produced gallium-68 (t 1/2 = 68 min, β em + = 89%) is used primarily to produce doses of PET radiopharmaceuticals from the local hospital radiopharmacy via a decentralised production model (Fig. 12). This is in contrast to the centralised production model exemplified by 18 F-radiopharmaceuticals like [ 18 F]FDG, which are produced in a large scale to serve several hospitals and imaging facilities from a single radiopharmaceutical production facility (Fig. 12). Gallium-68 metal-based radiochemistry is convenient and depending upon the age of the 68 Ge/ 68 Ga generator, can produce up to 3 patient doses per elution; increasing for high-capacity generators and cyclotron produced 68 Ga. Although some future developments may increase this capacity the shorter half-life of gallium-68 remains an issue for a centralised supply model. As we implement personalised medicine into routine clinical practice and use PET biomarkers to Page 20 of 28 Archibald and Allott EJNMMI radiopharm. chem. (2021) 6:30 underpin patient stratification and monitor treatment response, we need expedient solutions to scale radiopharmaceutical production to meet future clinical demand. The dose capacity of 68 Ge/ 68 Ga generators diminishes over time and is limited, which may be a concern if more 68 Ga-radiopharmaceuticals gain approval for routine clinical use. Additionally, while it has not been fully evaluated and is somewhat context dependent, the variable and diminishing molar activity (A m ) of 68 Ga over the lifespan of a generator may confound PET imaging of some biological targets. Finally, the availability of generators has been a challenge in recent years with long lead-times on delivery and rising cost, both of which tighten the already slim financial margin for operating a sustainable radiopharmacy (Mueller et al. 2019). To futureproof the throughput for producing a repertoire of 68 Ga-radiopharmaceuticals for the clinic, there is an option to commit resource to cyclotron produced gallium-68 where viable and effective methods have been described, producing > 3.5 GBq of 68 Ga within 60 min from a liquid target with a 14.3 MeV beam energy (Rodnick et al. 2020 (Hou et al. 2020). 2. Fluorine-18 radiochemistry is challenging! If we are to seek a centralised production model to produce 18 F-peptides previously labelled with 68 Ga, then a lot of development work is required. As we alluded to, developing fluorine-18 radiochemistry to radiolabel complex and sensitive molecules like proteins and peptides is challenging. The radiochemistry can be laborious and as the overall pharmacokinetic (PK) and metabolic profile is influenced by the radiolabelling and linker strategy, an iterative approach to developing suitable radioconjugates may be required which adds complexity. Several late-stage fluorination methods and 18 F-prosthetic group strategies suitable for radiolabelling peptides have been described and some evaluated in the clinic, but none as close in characteristics to the existing 68 Ga radiochemistry as the [ 18 F]AlF method (Allott et al. 2020;Narayanam et al. 2020;Kee et al. 2020;Yuan et al. 2018;Krishnan et al. 2017;Cole et al. 2014). Therefore, if the research experience lies with gallium-68 radiochemistry, the parallels offered by the [ 18 F]AlF method will be a facile adjustment.
Conventional fluorine-18 radiopharmaceuticals require automated radiosynthesis procedures for their GMP production, which adds additional complexity to the translational pathway (Allott and Aboagye 2020). However, automated procedures are convenient for [ 18 F]AlF-based radiopharmaceuticals and will be necessary where a centralized production model produces multi-patient doses for transportation to hospitals from a remote facility (Fig. 12). Automation is not a necessity for low level production runs to Page 21 of 28 Archibald and Allott EJNMMI radiopharm. chem. (2021) 6:30 access a couple of patient doses and therefore [ 18 F]AlF is also amendable to decentralised, manual kit-based production akin to 68 Ga-radiopharmaceuticals (Fig. 12) Ga-peptides have been developed in order to address the limitations of 68 Ga (i.e. short half-life and small dose batch sizes); but our opinion is that we should not consider [ 18 F] AlF-radiopharmaceuticals as a replacement for 68  Ga is, and always will be, a very important isotope that is unlikely to vanish from the clinic and actually, the flexibility that 68 Ga will afford the clinic is yet to come into its own. The more personalised treatments we implement, the greater range of imaging radiopharmaceuticals we will require to stage and monitor disease, treatment efficacy and progression. This will inevitably lead to a large toolbox of radiopharmaceuticals, some of which will be used every day-as we see currently with [ 18 F]FDG-where many patients benefit from a centralised production model which sets out to achieve multipatient and multihospital doses, allowing high patient throughput and in turn, lowers the cost-per-dose to healthcare services (Fig. 12); however, some radiopharmaceuticals may be used more infrequently, but their access is vital for patients with equivocal disease.
Therefore [ 18 F]AlF and 68 Ga should be considered as complementary labelling feedstocks that can streamline radiopharmaceutical production to meet demand so that imaging facilities have the best possible reach for their patient population and disease types. We have analogized this concept to a set of screwdrivers, where all are essential to perform the operation for which they are designed, but some are required more frequently than others; demand does not correlate with importance in specific disease detection (Fig. 13). With this in mind, we envisage that multidose [ 18 F]AlF radioconjugates will satisfy high demand imaging biomarkers and as a result, free up the Fig. 13 The screwdriver analogy. All tools are essential for the task they perform irrespective of how frequently that task is performed; the same is true for radiopharmaceuticals and therefore [ 18 F]AlF and 68 Ga radioconjugates both have an important role to play in modulating supply to meet the clinical demand for a range of imaging probes to offer patients the best possible care Page 22 of 28 Archibald and Allott EJNMMI radiopharm. chem. (2021) 6:30 radiopharmacy to produce small batches and a greater variety of 68 Ga radioconjugates for lower demand imaging biomarkers. We consider that the complementary nature of [ 18 F]AlF and 68 Ga radiochemistry and their marginal differences in PET scan quality will not devalue 68 Ga radiopharmaceuticals, but instead serves as a cohesive and powerful set of tools to improve the delivery of vital radiopharmaceuticals to the clinic.

The future of [ 18 F]AlF chelator design
We have an excellent variety of chelators to sequester [ 18 F]AlF (Fig. 3); however, further expansion of ambient temperature chelators with improved stability is an area of research that could be further developed. Interestingly, a computational approach to calculate the thermodynamic stability of zirconium-89 chelates has been reported and we propose that this approach could be used to prospectively optimise the design of [ 18 F] AlF chelators to focus synthesis efforts and experiment with underexplored functional groups (Holland 2020). The idea of developing more chelators can be supported or dismissed by the evaluation and clinical translation of [ 18 F]AlF-PSMA-11, which presents an interesting conundrum! The HBED chelator is a poor choice for [ 18 F]AlF chelation and results in a degree of in vivo demetallation; but, [ 18 F]AlF-PSMA-11 is being pursued clinically for prostate cancer imaging, a disease renowned for osteoblastic lesions where the potential for the mischaracterisation of metastatic disease by off-target bone uptake may be high; based on this, one may argue that we can "make do" with the chelators we already have. On the other hand, perhaps [ 18 F]AlF-PSMA-11 represents a scenario where we have placed the "horse before the cart" because the demand for [ 18 F] AlF-based PSMA radiopharmaceuticals is such that we are willing to take the path of least resistance and accept the limitations of sub-optimal chelation because the necessary radiochemistry precursors are commercially available at this moment in time, to support clinical studies. We don't think that this detracts from the implementation of optimal [ 18 F]AlF chelators in our radioconjugates, but rather supports the idea of rapid commercialisation of clinically important precursors for the community to implement and evaluate. With ever increasing interest in radionuclide therapy and theranostic isotope pairs, for example 68 Ga and 177 Lu which both form stable chelates with DOTA, should we now focus our attention to develop chelators to seamlessly support [ 18 F]AlF and 177 Lu isotope pairs from a single precursor molecule? There is a precedent for this approach in the literature (Lepage et al. 2020). Again, consideration needs to be given to future isotope requirements and, in this case, the impact of switching from an imaging isotope to a therapeutic isotope (where there may be future changes in demand).

Improving accessibility to [ 18 F]AlF-based radioconjugates
Automated methods are crucial if [ 18 F]AlF-based radiopharmaceuticals are to be implemented within a centralized production model Allott et al. 2017). Despite extensive work in developing a raft of [ 18 F]AlF-based radioligands for various targets, they are still not mainstream; an interesting communication by Hassan et al. (2019) discusses the challenge of implementing [ 18 F]AlF-based radiopharmaceuticals in 'normal' research institutions or university hospitals even when NOTA-peptide conjugates are commercially available. They correctly highlight that the expertise required Page 23 of 28 Archibald and Allott EJNMMI radiopharm. chem. (2021) 6:30 to implement the radiochemistry, and the manual nature [ 18 F]AlF-radiochemistry reserves these radiopharmaceuticals for well-trained radiochemists and radiopharmacists in dedicated research institutions (Hassan et al. 2019). In time to come, we will see the commercialization of cassettes to produce [ 18 F]AlF-radiopharmaceuticals on automated radiosynthesis platforms, simplifying the production of these products. This may not be the answer all production facilities are looking for, as automated radiochemistry required a large initial financial investment with full systems costing upwards of £100 k ($140 k), and continual maintenance and consumable overheads; however, automated radiochemistry has been significantly cost-reduced by the emergence of smaller platforms specifically designed for metal-based radiochemistry. Gallium-68 radioconjugates can now be produced from commercially available cassettes and we envisage that [ 18 F] AlF-radioconjugates will follow suit. If production scale is not a concern, then commercial and validated kits may provide a convenient solution. New technologies like microfluidic "lab-on-a-chip" devices which have been exemplified for metal-based radiochemistry may provide automated radiochemistry and quality control on a single, inexpensive device for clinical use (He et al. 2017(He et al. , 2016Zhang et al. 2020). These devices may also serve to improve the issues of fluoride concentration and efficiency of [ 18 F]AlF labelling (He et al. 2014;Wang and Dam 2020). While these are not fully established and are not yet commercially available, they may serve to bring safe, standardised and automated GMP production to facilities with less extensive infrastructure, staffed by nuclear medicine technologists.

Conclusions
The [ 18 F]AlF method has undoubtedly changed the landscape of fluorine-18 radiopharmaceutical development and adds another facet to metal-based radiolabelling that is relatively simple to implement and complements existing radiometal-based imaging agents. Over the last decade, the [ 18 F]AlF method has risen to the status of an effective tool in radiopharmaceutical design and has all the characteristic hallmarks of longevity (i.e. implementation in new radiopharmaceutical development projects for a range of disease phenotypes, automated methods, extensive translation into clinical trials); while we cannot predict the future landscape of PET with any certainty, we hope to see [ 18 F]AlF-based radiopharmaceuticals produced for the clinic on scale within the next 5-10 years.